This application relates to the treatment of foodstuffs using live cultures of protozoa, such as Tetrahymena thermophila. One aspect of the present invention is the provision of methods for the conversion of cholesterol present in foodstuffs into provitamin D3 and other sterols containing a double bond at position 7.
Animal milk is a complex mixture of different compounds, including lipids, proteins, minerals, sugars and vitamins (Russof, L. L. (1970), J. Dairy Science 53:1296-1302). The calcium, phosphate and vitamin D content of milk make it an adequate source of nutrients for bone formation (Fox, P. F. and McSweeney, P. L. H. (1998a) Salts of milk. In xe2x80x9cDairy Chemistry and Biochemistryxe2x80x9d, chapter 5, Blackie Academic and Professional, London). This may be a key aspect of its role in nature, allowing mammalian newborns to complete the formation of the skeleton after birth. Mineral and vitamin components of milk are also important to preserve bone structure in adulthood. Milk is also relatively economical, compared to other animal protein sources, and thus it makes a valuable contribution to the human diet (Russof, L. L. (1970), J. Dairy Science 53:1296-1302).
The lipid fraction of milk includes cholesterol, however, which has been implicated as a causative agent of coronary artery disease (Artaud-Wild, S. M., Connor, S. L., Sexton, G., Connor, W. E. (1993), Circulation 88:2771-2779). Other foodstuffs of animal origin such as eggs, which are commonly used in the preparation of a variety of food products, present the same problem. Because of the special organoleptic traits of milk and eggs, it is difficult to replace them by other products with less cholesterol content.
Patients with coronary heart disease (CHD) or hypercholesterolemia are commonly recommended to decrease their dietary cholesterol intake. Moreover, the general awareness of the risks associated with high blood cholesterol levels is an important factor limiting the consumption of eggs and dairy products by a health-conscious public. Cholesterol content is frequently indicated in the nutrition facts labels printed on food packages.
To address these problems, there is a need for methods to produce low-cholesterol versions of normally high-cholesterol foodstuffs, such as whole milk and eggs. Such methods should preferably not appreciably change the physical and organoleptic properties of the foodstuffs. The nutritional value of the treated foodstuffs should be preferably maintained, especially the levels of those components that are lipid-soluble and that are important for human nutrition (e.g., vitamins A and D, and essential fatty acids). Thus, the food treatment methods should yield products with lower cholesterol content but which are otherwise similar to the untreated foodstuffs. Additionally, the novel methods should preferably not require expensive equipment and materials or potentially toxic materials, such as organic solvents.
A number of methods have been described in patents in the US and other countries for reducing the cholesterol content of foodstuffs. For example, cholesterol can be removed from foodstuffs by the use of physicochemical methods. For instance, the use of supercritical fluids to produce liquid egg having reduced cholesterol content has been proposed (Ogasahara et al., U.S. Pat. No. 5,116,628). However, the high temperatures and pressures needed for the process can denature proteins present in the foodstuffs. Likewise, the production of low cholesterol butter oil by vapor sparging (Conte et al., U.S. Pat. No. 5,092,964) is another example of a method which, due to the extreme conditions used, is likely to denature proteins and alter organoleptic properties of the foodstuffs.
The use of organic solvents to extract cholesterol from foodstuffs has also been proposed. Thus, Fallis et al. (U.S. Pat. No. 4,104,286) have proposed the use of aqueous ethanol saponification and extraction with hydrocarbons and methanol to obtain free cholesterol, saponified fats and edible egg powder. This process uses extreme conditions and large quantities of organic solvents which may contaminate the processed foodstuffs. Extraction with liquid dimethylether (Yano et al., U.S. Pat. No. 4,234,619) is similarly inconvenient and does not appear to be selective for cholesterol as other neutral lipids are removed from the foodstuff. Johnson et al. (U.S. Pat. No. 4,997,668) applied solvent extraction to milk, but again the method does not appear to be selective for cholesterol and utilizes organic solvents which may contaminate foodstuffs.
A variation on the use of organic solvents is to employ oils to extract cholesterol from either aqueous or dry foodstuffs, like egg yolk and dairy products. (Bracco et al., U.S. Pat. No. 4,333,959; Keen, U.S. Pat. No. 5,039,541; Conte et al., U.S. Pat. No. 5,091,203; Merchant et al., U.S. Pat. No. 5,378,487; Jackeschky, U.S. Pat. No. 5,780,095). Again, these methods do not selectively extract cholesterol and oils contaminated with cholesterol are inevitably produced, which is undesirable.
Removal of cholesterol by formation of complexes with cyclodextrins has also been proposed for fatty substances of animal origin (Courregelongue et al., U.S. Pat. No. 4,880,573) and specifically in the case of dairy products (Chung Dae-Won, WO 9917620). The formation of complexes of cholesterol and saponin has also been described as a means to reduce cholesterol in milk (Richardson, U.S. Pat. No. 5,326,579). These methods are, however, too expensive for industrial applications.
A different approach is based on the use of enzymes that modify cholesterol. Thus, the use of cholesterol reductases, that modify cholesterol into poorly absorbed sterols, has been proposed (Beitz et al., U.S. Pat. No. 4,921,710; Ambrosius et al., U.S. Pat. No. 5,856,156). Another proposed enzymatic approach is the conversion of cholesterol into epicholesterol, which is then further modified by an epicholesterol dehydrogenase (Saito et al., U.S. Pat. No. 5,876,993). These methods are likely too expensive for industrial use, due to the cost of reasonably pure enzyme preparations, and they do not result in the conversion of cholesterol into useful compounds for human nutrition.
There is therefore a need for methods for treating foodstuffs to reduce the amount of cholesterol. Preferably, the cholesterol is converted to one or more substances that are useful for human nutrition, vitamin D, or a precursor of vitamin D such as the substances shown in FIG. 1. For example, as shown in FIG. 2, desaturation of cholesterol at position 7 converts it into provitamin D3, which upon UV irradiation in the skin can be activated to vitamin D3.
Additionally, there is a need for methods to increase the level of essential unsaturated fatty acids in milk. For example, gamma-linolenic acid (18:3, n-6) is a precursor of arachidonic acid, a second messenger molecule, which in turn is the source of many other important physiological compounds, like prostaglandins. The polyunsaturated fatty acid content of milk is relatively small compared to other fatty acids. Increasing the amount of n-6 unsaturated fatty acids in milk is desirable, because such modified milk can be a sufficient source of this type of essential fatty acid. Differences in coronary mortality can be explained by differences in cholesterol and saturated fat intakes in 40 countries but not in France and Finland (See, Artaud-Wild, S. M. et al., Circulation 88:2771-2779). Moreover, an enhanced level of this type of unsaturated fatty acid promotes cardiovascular health.
Most species of protozoa of the genuses Tetrahymena and Colpidium have no sterol nutritional requirement, as shown by their growth in the absence of exogenous sterols. Under such conditions, the major unsaponifiable components are tetrahymanol, a pentacyclic triterpenoid alcohol, and diplopterol, an isomer of tetrahymanol. Other minor components found are squalene and ubiquinone (Holz, G. G. and Conner, R. L. (1973) The composition, metabolism and roles of lipids in Tetrahymena. In xe2x80x9cBiology of Tetrahymenaxe2x80x9d (Elliot A M., ed). Dowden, Hutchinson and Ross, Stroudsberg, P., chapter 4, pp. 99-122; Wilton, D. C. (1983), Biochem. J. 216:203-206). When Tetrahymena is grown in the presence of exogenous sterols, however, the biosynthesis of tetrahymanol is completely inhibited and the added sterol is accumulated by the organism and, in most cases, metabolized to other sterols. Different types of biotransformations have been observed, including xcex947 and xcex9422 desaturation and the removal of ethyl, but not methyl groups, from C24 (Mallory, F. B. and Conner, R. L. (1971), Lipids 6:149-153; Conner, R. L., Landrey, J. R., Joseph, J. M., Nes, W. R., (1978) Lipids 13:692-696; Ferguson, K. A., Davis, F. M., Conner, R. L., Landrey, J. R. and Mallory, F. B. (1975), J. Biol. Chem. 250:6998-7005).
In the case of cholesterol, the biotransformations include conversion to xcex947,22-didehydrocholesterol, as the main derivative (a close analog of ergosterol, also called provitamin D2), together with xcex9422-dehydrocholesterol and xcex947-dehydrocholesterol (provitamin D3). The ratios between these desaturated derivatives can be controlled by changing culture conditions (Conner, R. L., Mallory, F. B., Landrey, J. R. and Iyengar, C. W. L. (1969), J. Biol. Chem. 244:2325-2333).
Further, Tetrahymena possesses a lipidic membrane rich in unsaturated fatty acids. The most abundant is gamma-linolenic acid (18:3, n-6). These organisms therefore have the potential to enhance the level of this type of essential fatty acid in the media where they grow. Moreover, they can synthesize these unsaturated fatty acids from saturated fatty acids, such as stearate and palmitate. A double benefit is thereby obtained: lowering the level of unwanted, saturated fatty acids, while increasing the level of desirable, unsaturated fatty acids. Further, Tetrahymena thermophila and T. pyriformis have been shown to produce and release to the surrounding medium, beta-galactosidase, an enzyme which splits lactose into glucose and galactose (Kiy, T. and Tiedtke, A. (1992), Appl. Microbiol. Biotechnol. 37:576-579). This enzyme may be useful for cleaving lactose in milk.
The present invention utilizes the foregoing properties of Tetrahymena, and other protozoa, to provide methods for treating cholesterol-containing foodstuffs (such as milk, egg yolk and other foodstuffs) to achieve one or more of the following goals: (a) reduce the level of cholesterol in foodstuffs; (b) reduce the level of saturated fatty acids in foodstuffs; (c) increase the level of unsaturated fatty acids in foodstuffs; (d) increase the level of one or more vitamin D precursors in foodstuffs and (e) reduce the level of at least some proteins in foodstuffs.
Thus, it is one object of the present invention to provide methods for reducing the amount of cholesterol in dairy products, such as milk, and in other cholesterol-containing foodstuffs, preferably without significantly altering the organoleptic properties of the treated foodstuffs.
It is another object of the present invention to provide methods for increasing the amount of xcex947-dehydrocholesterol (provitamin D3) and xcex947,22-didehydrocholesterol, which is a close analog of provitamin D2, in foodstuffs.
It is another object of the present invention to provide methods for increasing the amount of polyunsaturated fatty acids (PUFA) in cholesterol-containing foodstuffs (such as milk).
It is yet another aim of the present invention to proteolytically degrade selected proteins in foodstuffs, such as casein in milk and other dairy products.
One or more of the foregoing objectives can be accomplished equally, or each to a different extent, depending on the protozoan strain and cultivation conditions utilized.
In accordance with the foregoing, in one aspect the present invention provides methods for changing the composition of a cholesterol-containing foodstuff, the methods comprising the step of culturing a member of the family Tetrahymenidae in a liquid, cholesterol-containing foodstuff under conditions that enable one or more (or all) of the following changes in the composition of the foodstuff: (a) reduction in the level of cholesterol in the treated foodstuff; (b) reduction in the level of saturated fatty acids in the treated foodstuff; (c) increase in the level of unsaturated fatty acids in the treated foodstuff; (d) increase in the level of at least one vitamin D precursor in the treated foodstuff and (e) reduction in the level of at least one protein in the treated foodstuff. The foodstuff can be a dairy product, such as milk or egg yolk. The milk can be milk from any mammal, such as cow or goat, and egg yolk can be from eggs of any species of bird, such as domestic chicken eggs. Representative examples of other foodstuffs that can be treated by the methods of the present invention are broth and soups that contain materials of animal origin including cholesterol.
Optionally, the methods of the present invention include the additional step of removing substantially all of the cells of the family Tetrahymenidae from the liquid foodstuff after treatment of the liquid foodstuff. Examples of members of the family Tetrahymenidae useful in the practice of the present invention include Tetrahymena thermophila (T. thermophila), such as Tetrahymena thermophila strain CU399, and T. pyriformis, T. patula, T. rostrata, T. vorax, T. paravorax, T. chironomi, T. setifera, T. corlissi, T. stegomyiae and T. limacis. 
When the methods of the present invention are utilized to reduce the level of cholesterol in milk (such as whole milk), the level of cholesterol in the milk after treatment is preferably less than 50%, more preferably less than 20%, most preferably less than 5% of the level of cholesterol in the milk before treatment. Representative culture conditions for lowering the level of cholesterol in milk utilizing a member of the family Tetrahymenidae are: culture temperature of from 18xc2x0 C. to 38xc2x0 C., at a pH of between 5.0 and 8.0, for a time period of from one hour to 72 hours. Representative culture conditions for lowering the level of cholesterol in milk utilizing Tetrahymena thermophila are: culture temperature of from 24xc2x0 C. to 37xc2x0 C., at a pH of between 5 and 8, for a time period of from 1 hour to 72 hours.
In one embodiment of the invention, a member of the family Tetrahymenidae is cultured in a liquid, cholesterol-containing foodstuff under conditions that enable a reduction in the level of cholesterol in the treated foodstuff, and an increase in the level of at least one vitamin D precursor (formed by the biochemical conversion of the cholesterol to the vitamin D precursor(s)) in the treated foodstuff. Representative examples of vitamin D precursors are xcex947-dehydrocholesterol and xcex947,22-didehydrocholesterol. For example, the methods of the present invention can be used to increase the combined levels of xcex947-dehydrocholesterol and xcex947,22-didehydrocholesterol in milk to greater than 10 xcexcg/ml and 20 xcexcg/ml after 6 h and 12 h of treatment, respectively; or in other embodiments, to increase the combined levels of xcex947-dehydrocholesterol and xcex947,22-didehydrocholesterol in milk to greater than 20 xcexcg/ml and 45 xcexcg/ml after 6 h and 12 h of treatment, respectively.
In another embodiment of the invention, a member of the family Tetrahymenidae is cultured in a liquid, cholesterol-containing foodstuff under conditions that enable a reduction in the level of saturated fatty acids in the treated foodstuff and an increase in the level of polyunsaturated fatty acids in the treated foodstuff. In one embodiment, the content of xcex3-linolenic acid (18:3 (n-6)), in treated milk increases between 2 to 6 fold, from about 2 mg/100 g milk in untreated milk to about 12 mg/100 g milk in milk treated with Tetrahymena for 12 hours.
In another embodiment of the invention, a member of the family Tetrahymenidae is cultured in a liquid, cholesterol-containing foodstuff under conditions that enable a reduction in the level of casein in the treated foodstuff. For example, in one embodiment, the methods of the present invention can be used to reduce the level of casein in milk to less than 70%, preferably less than 40%, more preferably less than 10%, of the level of casein in the milk before treatment.
The methods of the present invention provide several advantages. For example, in some embodiments the methods not only selectively decrease foodstuff cholesterol concentration, preferably without significantly altering organoleptic properties, but at the same time, the process of desaturating cholesterol at position 7, converts it into xcex947-dehydrocholesterol, also known as provitamin D3. Thus, a single reaction decreases an unwanted material (cholesterol), converting it into a desirable one (provitamin D3). Moreover, in some embodiments, the methods of the invention enhance the contents of essential unsaturated fatty acids, especially 18:3 (n-6) fatty acids, reported to possess cardiovascular health promoting properties. Also, the proteolytic breakdown of proteins, such as milk casein, which may have undesirable effects, can be achieved utilizing some embodiments of the inventive methods described herein.